PDK1 regulates FA disassembly on vitronectin. (A,B) Control (shScrl) and PDK1 knockdown (shPDK1_79 and 81) endothelial cells transfected with Paxillin–GFP (green) that had been seeded on vitronectin, were treated with nocodazole (t = 0 min NOCO washout, A). Then the drug was washed out (t = 30 min NOCO washout; B) and cells were stained with anti-α tubulin (red) and anti-αv (magenta, DAPI in blue). Scale bars: 20 µm. (C) Quantification of FA area/cell and average FA size. Data were plotted as the mean±s.d.; °P<0.001, ^†P<0.05 for knockdown endothelial cells versus control endothelial cells.

PDK1 transiently localises to FAs and associates with αvβ3 integrin. (A,B) Endothelial cells that had been seeded on vitronectin and transfected with paxillin–GFP (green), were treated with nocodazole (t = 0 min NOCO washout, A). Then the drug was washed out (t = 30 min NOCO washout, B) and cells were stained with anti-αv integrin (turquoise) and anti-PDK1 (red, DAPI in blue). The merged images originate from the overlap of paxillin and PDK1 staining; arrows indicate zoomed-in regions. Scale bars: 10 µm. (C) Endothelial cells that had been seeded on vitronectin and transfected with paxillin–GFP (green), were fixed after 2 hours of adhesion and stained with anti-αvβ3 integrin (turquoise) and anti-PDK1 (red, DAPI in blue). The merged images originate from the overlap of paxillin and PDK1 fluorescence; arrows indicate the zoomed regions. Scale bars: 20 µm. (D) Empty vector-infected (−) or PDK1-overexpressing (+) endothelial cells were treated with nocodazole (t = 0 min NOCO wash). After nocodazole removal (t = 30 min NOCO wash), endothelial cells were lysed and αvβ3 was immunoprecipitated; immunocomplexes and corresponding lysates were immunoblotted with the indicated antibody. (E) Wild-type endothelial cells were lysed and αvβ3 was immunoprecipitated; immunocomplexes and corresponding lysate were immunoblotted with the indicated antibody. (F) Control (shScrl) and PDK1 knockdown (shPDK1_79 and 81) endothelial cells were lysed and αvβ3 was immunoprecipitated; immunocomplexes and corresponding lysates were immunoblotted with the indicated antibody. (G) Wild-type endothelial cells were lysed and pull-down assays were carried out using the cytoplasmic tail of β3 integrin fused to GST; immunocomplexes and corresponding lysate were immunoblotted with the indicated antibody. (H) Endothelial cells infected with empty vector (−) or overexpressing PDK1 (+) were lysed and peptide pull-down assays were carried out using a synthetic peptide corresponding to the cytoplasmic part of αv and α2 integrin. Western blots shown are representative of four experiments performed with similar results. (I) Endothelial cells were seeded on vitronectin and then analysed in PLAs with the following antibodies: anti-PDK1 and anti-αv (PDK1+αv), anti-PDK1 and anti-β3 (PDK1+β3), anti-PDK1 and anti-α3 (PDK1+ α3) and anti-PDK1 and rabbit IgG (PDK1+rabbit IgG) as a negative control. DAPI is in blue, red spots represent PLA signals. Scale bars: 20 µm for PDK1+rabbit IgG, 50 µm for other images.

PDK1 controls αvβ3 endocytosis during FA disassembly. (A) Control (shScrl) and PDK1 knockdown (shPDK1_79 and 81) endothelial cells were plated on vitronectin and incubated with anti-β3 antibody either at 4°C as control or at 37°C in presence of primaquine to allow internalisation of antibody-bound integrin (red, DAPI in blue); as a control of β3 surface staining, acid wash was done or not. Scale bars: 50 µm. (B) Quantification of the total vesicle area/cell after 10, 20 and 30 minutes of endocytosis. Data were plotted as the mean±s.d.; ^§P<0.005 for knockdown endothelial cells versus control endothelial cells. (C) Endocytosis of αvβ3 in presence of primaquine was evaluated in control (shScrl) and PDK1 knockdown (shPDK1_79 and 81) endothelial cells after 10, 20 and 30 minutes. Internalised biotinylated integrin was quantified by ELISA. Data were plotted as mean±s.d. of the percentage of total biotinylated integrin αvβ3 that had been internalised; ^§P<0.005 for knockdown endothelial cells versus control endothelial cells. (D–F) Control (shScrl) and PDK1 knockdown (shPDK1_79 and 81) endothelial cells that had been seeded on vitronectin, were treated with nocodazole (t = 0 min). Then, during the drug washout (t = 30 min), cells were incubated with anti-β3 antibody in presence of primaquine and internalisation of antibody-integrin complexes were followed by indirect immunoflourescence. FA disassembly was confirmed with anti-paxillin antibody (magenta, internalised β3 integrin in green, DAPI in blue). Scale bars: 50 µm. (G) Quantification of vesicle area/cell. Data were plotted as mean±s.d.; ^†P<0.05 for knockdown endothelial cells versus control endothelial cells. (H) Control (shScrl) and PDK1 knockdown (shPDK1_79 and 81) endothelial cells were treated with nocodazole and αvβ3 endocytosis was followed during drug washout with or without primaquine. Internalised biotinylated integrin was quantified by ELISA. Data were plotted as the mean±s.d. of the percentage of total biotinylated αvβ3 integrin that had been internalised; ^§P<0.005 for knockdown endothelial cells versus control endothelial cells.

PDK1 needs both the PH domain and kinase activity to control αvβ3 integrin endocytosis. (A) shPDK1_79 endothelial cells infected with lentivirus carrying different PDK1 mutants resistant to silencing (PDK1-wt, PDK1-caax, PDK1-ΔPH, PDK1-KD) and control endothelial cells (shScrl) were plated on vitronectin and incubated with anti-β3 antibody in presence of primaquine to allow internalisation of antibody-bound integrin (red, DAPI in blue). Scale bars: 20 µm. (B) Quantification of vesicle area/cell of experiment shown in A. Data were plotted as the mean±s.d.; ^†P<0.05 for knockdown endothelial cells versus control endothelial cells. (C) endothelial cells were transfected with Vinculin–RFP (red) in combination with different YFP-fused mutants of PDK1 (wt, ΔPH and KD, in green), seeded on vitronectin and fixed after 2 hours of adhesion; arrows indicate the zoomed regions. Scale bars: 20 µm. (D) Endocytosis of β3 and Tfn-555 was performed as described in A with wild-type endothelial cells in presence of Akt inhibitor (Akti), LY294002 or vehicle; then total vesicle area/cell was quantified. Data were plotted as mean±s.d.; ^†P<0.05 for treated cells versus control endothelial cells.

The cytoplasmic tail of β3 integrin is phosphorylated by PDK1. (A) HEK 293T cells expressing GST-β3 cyto were treated for 1 hour with either the PDK1 inhibitor BX-795 (PDKi) or calyculin A (Cal); then lysed and purified with Glutathione Sepharose; GST-proteins were immunoblotted with the indicated antibody. (B) endothelial cells treated for 1 hour with the PDK1 inhibitor BX-795 (PDKi) were lysed and αvβ3 was immunoprecipitated; immunocomplexes and corresponding lysates were immunoblotted with the indicated antibody. (C) Endothelial cells that had been seeded on vitronectin, were treated with either the PDK1 inhibitor BX-795 (PDKi) or Akt inhibitor (AKTi) for 1 hour and a PLA assay was performed with the following antibodies: anti-β3/anti-pSer/Thr Akt-substrate. DAPI is in blue, red spots represent PLA signals. Scale bars: 50 µm. (D) Control (shScrl) and PDK1 knockdown (shPDK1_79 and 81) endothelial cells were lysed and αvβ3 was immunoprecipitated; immunocomplexes were immunoblotted with the indicated antibody. (E) β3 wild-type- and β3 T753A-expressing endothelial cells that had been seeded on vitronectin, were fixed after 2 hours of adhesion and stained with anti-phospho FAK (green) and anti-FLAG (red, DAPI in blue). Scale bars: 20 µm. (F) β3 wild-type- and β3 T753A-expressing endothelial cells were lysed and β3 was immunoprecipitated; immunocomplexes were immunoblotted with the indicated antibody.

Thr753 of β3 integrin regulates its endocytosis. (A,B) Endocytosis of β3-FLAG in presence of primaquine was evaluated in β3 wild-type- and β3 T753A-expressing endothelial cells after 10, 20 and 30 minutes. Total (tot) and internalised biotinylated integrin was quantified by streptavidin-agarose immunoprecipitation and anti-FLAG immunoblotting (A). Data were plotted as the mean±s.d. of the percentage of total biotinylated β3-FLAG integrin that had been internalised (B); ^†P<0.05 for β3 T753A- endothelial cells versus β3 wild-type endothelial cells. (C) β3 wild-type- and β3 T753A-expressing endothelial cells were plated on vitronectin and incubated with anti-β3 antibody at 37°C in presence of primaquine to allow internalisation of antibody-bound integrin (green, DAPI in blue); as a control ectopic β3 integrin was stained with anti-FLAG antibody (red). Scale bars: 50 µm. (D) Quantification of total vesicles area/cell after 30 minutes of endocytosis. Data were plotted as the mean±s.d.; ^†P<0.05 for β3 T753A-expressing endothelial cells versus β3 wild-type-expressing endothelial cells.